Abstract
Urinary tract infections (UTI) are among the most frequently encountered infections in clinical practice globally. Predominantly a burden among female adults and infants, UTIs primarily caused by uropathogenic Escherichia coli (UPEC) results in high morbidity and fiscal health strains. During pathogenesis, colonization of the urinary tract via fimbrial adhesion to mucosal cells is the most critical point in infection and has been linked to DNA methylation. Furthermore, with continuous exposure to antibiotics as the standard therapeutic strategy, UPEC has evolved to become highly adaptable in circumventing the effect of antimicrobial agents and host defenses. Hence, the need for alternative treatment strategies arises. Since differential DNA methylation is observed as a critical precursor to virulence in various pathogenic bacteria, this body of work sought to assess the influence of the DNA adenine methylase (dam) gene on gene expression and cellular adhesion in UPEC and its potential as a therapeutic target. To monitor the influence of dam on attachment and FQ resistance, selected UPEC dam mutants created via one-step allelic exchange were transformed with cloned qnrA and dam complement plasmid for comparative analysis of growth rate, antimicrobial susceptibility, biofilm formation, gene expression, and mammalian cell attachment. The absence of DNA methylation among dam mutants was apparent. Varying deficiencies in cell growth, antimicrobial resistance and biofilm formation, alongside low-level increases in gene expression (recA and papI), and adherence to HEK-293 and HTB-9 mammalian cells were also detected as a factor of SOS induction to result in increased mutability. Phenotypic characteristics of parental strains were restored in dam complement strains. Dam’s vital role in DNA methylation and gene expression in local UPEC isolates was confirmed. Similarly to dam-deficient Enterohemorrhagic E. coli (EHEC), these findings suggest unsuccessful therapeutic use of Dam inhibitors against UPEC or dam-deficient UPEC strains as attenuated live vaccines. However, further investigations are necessary to determine the post-transcriptional influence of dam on the regulatory network of virulence genes central to pathogenesis.
Highlights
Uropathogenic Escherichia coli (UPEC) is the most ubiquitous pathogen implicated in urinary tract infections (UTIs), accounting for 80–90% of all infections [1, 2]
Of the 174 FQ-R resistant isolates including cured uropathogenic Escherichia coli (UPEC) strains A620b, C119, U155, CFT073, and MG1655 screened, 71% were positive for the dam sequence amplified by UR427/ UR428 to produce a 1071 bp product (Figure 1B)
To assess the putative role of dam on virulence, dam-deficient mutant strains were successfully constructed using Red-recombinase-mediated allelic exchange as the wild-type dam gene was interrupted with a chloramphenicolresistance gene (Figure 1A)
Summary
Uropathogenic Escherichia coli (UPEC) is the most ubiquitous pathogen implicated in urinary tract infections (UTIs), accounting for 80–90% of all infections [1, 2]. Several publications report the existence of these virulence factors in clusters, small virulence cassettes, or large blocks of genes in enteropathogenic, enterohaemorragic, and UPEC strains, not observed in the genome of fecal isolates [3, 8,9,10,11,12,13,14] These factors are often linked or co-regulated, acting in concert according to host response and environmental signals [15]. The pap operon mainly consists of fimbriae structural subunits (papA, -C, -D, -E, -F, -G); subunit to terminate fimbrial growth and anchor mature fimbriae to host cell surface, papH; and the divergently encoded regulatory genes papB and papI, within which the main promoter is located. There are reports that support fimbriae-mediated UPEC adherence in vitro [7, 23, 24], the significance of P fimbriae for infectivity has not been confirmed as only subtle adherence roles were exhibited in uroepithelial cell culture models [25]
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